Polarized epithelial cells line every organ and are of utmost importance for the function of the human body. Central to epithelial cell function is the establishment and maintenance of biochemically and functionally distinct membrane domains, the apical membrane facing the lumen of an organ and the basolateral membrane that is in contact with connective tissues. Both membranes are separated by tight junctions. To maintain this architecture, epithelial cells must continuously sort newly synthesized and internalized transmembrane receptors to the correct membrane domains in the biosynthetic and endocytic pathways. Our work focuses on the molecular mechanisms that ensure correct targeting to the basolateral membrane from a central sorting station, the recycling endosomes. Previously we showed that cargos destined for the basolateral membrane are recognized by the epithelial-specific clathrin adaptor complex AP-1B for incorporation into AP-1B vesicles. This grant application proposes to follow up on these findings and to investigate the molecular mechanisms of AP-1B function, membrane recruitment, and regulation of the AP-1B-dependent pathway from recycling endosomes to the basolateral membrane. We will employ genetical, biochemical, and cell biological methods to accomplish our research goals. PUBLIC HEALTH RELEVANCE: The primary function of epithelial cells is to ensure the correct nutrient and waste product exchange between the body and the environment. To fulfill these different functions, the surface of epithelial cells is divided into biochemically and functionally distinct membrane domains. Our long-term goal is to understand how polarized epithelial cells establish and maintain this asymmetry with a focus on processes that lead to correct targeting of transmembrane receptors to the membrane domain that faces connective tissues and neighboring cells.